Delivery of dsRNA to arthropods

ABSTRACT

The invention is to methods of gene silencing in arthropods using dsRNA. The method is include contacting the arthropod with, and/or directly feeding the arthropod, the dsRNA to the arthropods to deliver the dsRNA to arthropod tissues. It is envisaged that the methods of the invention will have use in determining the biological function of genes in arthropods. Methods of pest control of arthropods, and of protecting arthropods against parasites and predators are provided. Transgenic arthropods expressing dsRNA molecules are also provided by the present invention.

This application is a continuation of U.S. Ser. No. 10/482,888, filedJun. 14, 2004 now U.S. Pat. No. 8,101,343 as a §371 national stage ofPCT International Application No. PCT/AU02/00897, filed Jul. 5, 2002,which claims priority of Australian Application No. PR6215, filed Jul.6, 2001, each of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The Present Invention Relates Generally to dsRNA and its Use in Genesilencing. Furthermore, the present invention relates to methods ofdelivering dsRNA to an arthropod.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is considered as a naturally occurring adaptivedefense in at least some organisms against viruses and the production ofaberrant transcripts, such as those produced by transposon mobility(Bosher and Labouesse, 2000; Waterhouse et al., 2001).

The actual process by which dsRNA mediates target RNA degradation is notfully understood, but the cellular machinery involved is gradually beingidentified. Full-length dsRNAs have been observed to be progressivelydegraded into ˜21-nucleotide dsRNAs, by an enzyme called Dicer-1(Elbashir et al., 2001). It is believed that the Dicer-1 proteins, alongwith their associated 21-mer dsRNA, seek single stranded RNAs withsequence identity, and promote the cleavage of single stranded RNAtargets (Waterhouse et al., 2001).

The intestine of C. elegans is a simple tube constructed of only 20cells (White, 1988). For C. elegans, dsRNA has been microinjected intothe gonadal tissues of adult worms, but simpler methods have since beendeveloped that circumvent the tedious microinjection method. Nematodesfed Escherichia coli bacteria that simultaneously express sense andantisense RNAs can acquire dsRNA. Interestingly, the ingested dsRNA canthen spread from the gut to target almost all tissues with the nematode(Timmons and Fire, 1998). Alternatively, the worms can be soaked indsRNA solutions, either with liposomes or as naked RNA (Tabara et al.,1998; Maeda et al., 2001).

Arthropod guts are comprised of a great many cell types, and are highlyvariable, as they have adapted to the needs of each species and theirunique dietary choices. The evolutionary distance between nematodes andinsects is considerable, and there is no reason to assume that whilefeeding dsRNA to C. elegans was successful, it would be a techniqueeasily transferable to insects. The presence of specific barriers ininsect guts, such as the peritrophic membrane, could also limit orprevent direct absorption of orally delivered dsRNA. The midgut of anarthropod is the primary site of nutrient uptake, and midgut internalenvironments of different arthropods can vary widely. For example, thefruit fly Drosophila melanogaster has a rather acidic midgut lumen,while many Lepidoptera (moths and butterflies) have a very hostile,highly basic midgut environment.

SUMMARY OF THE INVENTION

The present invention provides methods that utilize dsRNA to determinethe biological function of an RNA in an arthropod. In particular, theinvention provides efficient mechanisms of delivering dsRNA to anarthropod with the aid of transfection promoting agents. Furthermore,the present invention provides methods for controlling pest arthropodpopulations, methods for controlling pathogens carried by arthropods, aswell as methods for protecting an arthropod from a pathogen, parasite orpredatory organism. In addition, the present invention providestransgenic organisms, in particular arthropods, expressing small dsRNAmolecules.

In one aspect, the present invention provides a method of determiningthe biological function of a target RNA in an arthropod comprisingdelivering to the arthropod a dsRNA molecule which specifically reducesthe level of the target RNA and/or the production of a protein encodedby the target RNA in a cell of the arthropod, and assessing the effectof the dsRNA on at least one biological function of the arthropod.

The method of the present invention can be utilized to rapidly screenuncharacterized RNAs or expressed sequence tags (ESTs) for afunction(s), particularly in high(er) throughput screens of pestarthropod EST libraries. Ultimately, the method facilitates theidentification of novel pesticide targets. For example, a particulardsRNA that confers lethality on an arthropod indicates that thecorresponding RNA itself, or the protein encoded by a mRNA, is essentialfor arthropod survival, and, as a consequence, said RNA or protein is agood pesticide target. Accordingly, this RNA, or the protein encoded bythe mRNA, is specifically targeted in the design of, and/or, screeningfor, agents to control pest populations of the arthropod.

In an alternative embodiment, the method is used to determine anyadditional function(s) of previously characterized arthropod RNAs.

Alternatively, dsRNA is designed with specificity to an RNA that ispotentially involved in particular biological processes (for example,determined by sequence identity with known genes, and/or throughexpression patterns) and can be screened to obtain a dsRNA that producesa particular phenotype. Such phenotypes include arthropod death orsterility. In fact, random dsRNA can be screened by this method for adesired phenotype.

Pathogens, such as viruses, which infect arthropods can be engineered toexpress a dsRNA for the down-regulation of a specific RNA. Typically,this would be for the production of biological agents to control a pestpopulation of arthropods. However, such pathogens may not be easilymanipulated, slowing down the progress of identifying suitablegenetically engineered pathogens. The present invention can be used torapidly screen candidate dsRNA molecules to determine if they producethe desired effect on a target arthropod pest. Once a candidate has beenshown to produce the desired effect, suitable pathogens can beengineered and tested as biological control agents of an arthropodpopulation.

The method of the invention can also be used to identify RNA importantfor enhancing production traits of an arthropod. In this instance, theactivity of the dsRNA can down-regulate the production trait. Onceidentified, the relevant genes can be overexpressed to enhance theseproduction traits. In accordance with this embodiment of the invention,the corresponding endogenous arthropod gene is ectopically expressed inthe arthropod to enhance the production trait. Exemplary productiontraits contemplated herein include the composition and/or quantity ofhoney produced by bees, and the growth rate and/or size of ediblecrustaceans such as prawns, crayfish and lobsters, and the like.

In an alternate use of the method of the present invention, a target RNAcan be assessed to determine whether it, or a protein encoded by theRNA, is acted upon by an agent such as a pesticide. In this instance,the method also comprises exposing the arthropod to the agent, whereinif the agent has little or no additional effect on the arthropod itindicates that the RNA, or protein encoded by the RNA, is directly actedupon by the agent or is involved in a biological pathway which iseffected by the agent. Upon the identification of the mechanism ofaction of the agent, this information can be used to design alternatepesticides (for example) which act on the same molecules/pathways. Thisis particularly useful where an agent is known to be a potent pesticide,however, it is not approved for use due to concerns such as its toxicityto non-pest organisms.

In a preferred embodiment, the dsRNA is delivered by a processcomprising contacting the arthropod with the dsRNA. Preferably, saidcontacting comprises wholly or partially soaking the arthropod in acomposition comprising the dsRNA.

In a further preferred embodiment, the dsRNA is delivered by a processcomprising feeding the dsRNA to the arthropod.

Preferably, the dsRNA is delivered in a composition comprising atransfection promoting agent. More preferably, the transfectionpromoting agent is a lipid-containing compound.

In one embodiment, the lipid-containing compound is selected from thegroup consisting of; Lipofectamine, Cellfectin, DMRIE-C, DOTAP andLipofectin. In another embodiment, the lipid-containing compound is aTris cationic lipid. Examples of suitable Tris cationic lipids include,but are not limited to, CS096, CS102, CS129, CS078, CS051, CS027, CS041,CS042, CS060, CS039, or CS015.

Preferably, the composition further comprises a nucleic acid condensingagent. The nucleic acid condensing agent can be any such compound knownin the art. Examples of nucleic acid condensing agents include, but arenot limited to, spermidine (N-[3-aminopropyl]-1,4-butanediamine),protamine sulphate, poly-lysine as well as other positively chargedpeptides. Preferably, the nucleic acid condensing agent is spermidine orprotamine sulfate.

In yet another preferred embodiment, the composition further comprisesbuffered sucrose or phosphate buffered saline.

In an alternate embodiment, the dsRNA is delivered by a processcomprising feeding a transgenic organism expressing the dsRNA to thearthropod. The transgenic organism is selected from, but not limited to,the group consisting of: plants, yeast, fungi, algae, bacteria oranother arthropod expressing the dsRNA. Examples of suitable bacteriainclude Pseudomonas fluorescens, E. coli, B. subtilis (Gawron-Burke andBaum, 1991), and Wolbachia sp. Preferably, the transgenic organism is atransgenic plant.

In yet another embodiment, the dsRNA is delivered by a processcomprising contacting the arthropod with a virus expressing the dsRNA.

Preferably, the dsRNA comprises a nucleotide sequence having at least90% identity to at least a portion of the sequence of the target RNA,more preferably the dsRNA comprises a nucleotide sequence having atleast 97% identity to at least a portion of the sequence of the targetRNA, and even more preferably the dsRNA comprises a nucleotide sequencehaving at least 99% identity to at least a portion of the sequence ofthe target RNA.

The dsRNA has a region of self-complementarity to permit it assuming adouble-stranded conformation in an arthropod host. Preferably, theregion of self-complementary corresponds to at least about 20 to about23 contiguous nucleotides of the target RNA, more preferably the fulllength sequence of the target RNA.

The arthropod can be any species. Preferably, the arthropod is ofeconomic importance, such as, for example, an edible crustacean, anarthropod that causes disease, a household pest, an agricultural pest,or an arthropod that produces a useful substance or compound, such as,for example, silk, an edible substance (e.g. honey) or a medicinalsubstance or compound (e.g. a toxin or venom).

It is preferred that the arthropod is an insect or a crustacean. Mostpreferably the arthropod is an insect.

The arthropod can be at any stage of development, however, it ispreferred that the arthropod is in a larval or adult developmental stagewhen the dsRNA is delivered. The present invention clearly encompassesdetermining the effect of the dsRNA on a phenotype of the arthropod at alater development stage even when the dsRNA is delivered at an earlierdevelopmental stage.

Preferably, the RNA is mRNA.

In a further embodiment, the dsRNA molecule is designed based on thenucleotide sequence of an EST that has been derived from mRNA isolatedfrom the arthropod.

In another aspect, the present invention provides a compositioncomprising dsRNA and a transfection promoting agent, wherein said dsRNAcomprises a nucleotide sequence that it is at least 90% identical to thesequence of a target RNA, wherein the target RNA is selected from thegroup consisting of: a naturally-occurring arthropod RNA, anaturally-occurring RNA of an organism that is a pathogen carried by anarthropod, a naturally-occurring RNA of a virus that infects anarthropod, an RNA copy of a naturally-occurring DNA virus that infectsan arthropod, and a naturally-occurring RNA of a bacterium that infectsan arthropod.

It is preferred that the naturally occurring arthropod RNA is an mRNAwhich encodes a protein involved in, and more preferably essential for,arthropod development, neural function, reproduction or digestion.

Preferably, the transfection promoting agent is a lipid-containingcompound.

In one embodiment, the lipid-containing compound is selected from thegroup consisting of; Lipofectamine, Cellfectin, DMRIE-C, DOTAP andLipofectin. In another embodiment, the lipid-containing compound is aTris cationic lipid. Examples of suitable Tris cationic lipids include,but are not to limited to, CS096, CS102, CS129, CS078, CS051, CS027,CS041, CS042, CS060, CS039, or CS015.

Preferably, the composition further comprises a nucleic acid condensingagent. The nucleic acid condensing agent can be any such compound knownin the art. Examples include, but are not limited to, spermidine(N-[3-aminopropyl]-1,4-butanediamine), protamine sulphate, poly-lysineas well as other positively charged peptides. Preferably, the nucleicacid condensing agent is spermidine or protamine sulfate.

Preferably, the composition is formulated such that it can be applied toan area inhabited by a population of arthropods. This area can includecrop plants, ornamental or native plants, or animals. Furthermore, thecomposition can be applied directly to an animal such as a cow or asheep. Accordingly, in a preferred embodiment the composition furthercomprises an agriculturally acceptable carrier.

The composition of the present invention can also be formulated as abait. In this instance, the composition further comprises a foodsubstance and/or an attractant, such as a pheromone, to enhance theattractiveness of the bait to the arthropod.

In a further aspect, the present invention provides a method ofcontrolling an arthropod pest comprising delivering to the arthropoddsRNA by a process comprising contacting the arthropod with said dsRNAor feeding said dsRNA to the arthropod, for a time and under conditionssufficient for said dsRNA, or a degradation product thereof, tospecifically reduce the level of a target RNA and/or the production of aprotein encoded by the target RNA in a cell of the arthropod, whereinthe target RNA or the protein is important for arthropod survival,development and/or reproduction.

Preferably, the dsRNA is delivered in a composition according to theinvention.

Preferably, the target RNA or the target protein is essential forarthropod development, neural function, reproduction or digestion.

The present invention is also used to control disease pathogens carriedby arthropods. For instance, there are ecological arguments for notdestroying mosquitoes to control malaria, sleeping sickness, and manyarboviruses.

Accordingly, in yet another aspect, the present invention provides amethod for controlling a pathogen transmitted by an arthropod, themethod comprising delivering to the arthropod dsRNA by a processcomprising contacting the arthropod with said dsRNA or feeding saiddsRNA to said arthropod, for a time and under conditions sufficient forsaid dsRNA, or a degradation product thereof, to specifically reduce thelevel of a target RNA and/or the production of a protein encoded by thetarget RNA in a cell of the pathogen, wherein the target RNA or theprotein is important for pathogen survival, development and/orreproduction.

Preferably, the dsRNA is delivered in a composition according to theinvention.

In preferred embodiment of the third aspect, the pathogen is selectedfrom the group consisting of fungi, protozoans, bacteria and viruses.

In the instance where the pathogen is a virus, the presence of thedsRNA, or degradation products thereof, in a cell of the arthropodspecifically reduces the accumulation of a target RNA or the productionof a protein essential for viral survival and/or replication.

Beneficial arthropods can be protected from parasite/pathogen attack bythe delivery of appropriate dsRNA containing compositions. Insectcolonies, in particular those such as bees, silkworms, or evenlaboratory stocks of insects, can be protected from parasitic orpredatory pests (eg. nematodes, mites), or viral and microbialpathogens. Similarly, commercially important stocks of crustaceans canbe protected from disease pathogens.

Thus, in a further aspect the present invention provides a method ofprotecting an arthropod against a pathogen, parasite or predatoryorganism, the method comprising delivering to the arthropod dsRNA by aprocess comprising contacting the arthropod with said dsRNA or feedingsaid dsRNA to said arthropod, for a time and under conditions sufficientfor said dsRNA, or a degradation product thereof, to specifically reducethe level of a target RNA and/or the production of a protein encoded bythe target RNA in a cell of the pathogen, parasite or predatoryorganism, wherein the target RNA or the protein is important for thesurvival, development and/or reproduction of the pathogen, parasite orpredatory organism.

Preferably, the dsRNA is delivered in a composition according to theinvention.

In the instance where the pathogen is a virus, the presence of thedsRNA, or degradation products thereof, in a cell of the arthropodspecifically reduces the accumulation of RNA or the production of aprotein essential for viral survival and/or replication.

Previously, dsRNA techniques have involved the use of constructs inwhich the dsRNA approximates the length of the entire open reading frameof a RNA or a substantial portion thereof. The present inventors havefound that such long dsRNA constructs are not required in order toobtain RNA interference. Surprisingly, the present inventors have foundthat dsRNA as little as 21 nucleotides are capable of gene silencing.Furthermore, the present inventors have also surprisingly found thatdsRNA that had been previously processed and partially degraded withinone organism can still facilitate RNAi in another arthropod.

Hence, in another aspect the present invention provides a transgenicorganism comprising a heterologous nucleic acid(s) which is transcribedto produce a dsRNA, wherein the portion of the dsRNA which is doublestranded is about 21 to about 50 base pairs in length.

Preferably, the dsRNA comprises a nucleotide sequence having at least90% identity to at least a portion of the sequence of a target RNAselected from the group consisting of: a naturally-occurring arthropodRNA, a naturally-occurring RNA of an organism that is a pathogen carriedby an arthropod, a naturally-occurring RNA of a virus that infects anarthropod, an RNA copy of a naturally-occurring DNA virus that infectsan arthropod, and a naturally-occurring RNA of a bacterium that infectsan arthropod.

Preferably, the portion of the dsRNA which is double stranded is about21 to about 23 base pairs in length.

Preferably, the organism is selected from the group consisting of:plants and arthropods.

In the instance where the transgenic organism is a plant, the dsRNA ispreferably at least 90% identical to at least a portion of a RNAexpressed in an arthropod which feeds on the plant.

Preferably, the dsRNA increases the resistance of the transgenicorganism to a pathogen. Preferably, the pathogen is a virus.

Preferably, the dsRNA is produced as a single open reading frame in thetransgenic organism, where the sense and anti-sense sequences areflanked by an unrelated sequence which enables the sense and anti-sensesequences to hybridize to form the dsRNA molecule with the unrelatedsequence forming a loop structure.

As will be apparent, preferred features and characteristics of oneaspect of the invention can be applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

The invention will hereinafter be described by way of the followingnon-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. PCR detection of phspGUS[i/r] plasmid in insects injected withthe plasmid as late embryos. In the leftmost 7 lanes, the presence ofthe GUS transgene in the flies is evident by the production of the 1 kbPCR product in all developmental stages. There was no PCR product inwild type, non-transgenic flies (wt). In the right hand side of the gel,only L1 (1^(st) instar larvae) show evidence of the injected plasmid, asindicated by the single 500 bp PCR product.

FIG. 2. Gene silencing in D. melanogaster larvae and adults afterfeeding neonates dsRNA. Neonate larvae were soaked in a compositioncomprising transfection promoting agent and GUS dsRNA, and individualswere assayed either as 2^(nd) instar larvae (top panel) or as adults(bottom panel). A total of 40 individuals were assayed for each group.Each dot represents one individual's level of GUS gene silencing,relative to non-treated controls

FIG. 3. Reduced GUS activity following soaking of neonate larvae in acomposition comprising transfection promoting agent and differentconcentrations of dsRNA. Each dot represents one individual adult fly'sGUS activity, as a percentage of non-treated GUS controls. A total of 20flies were assayed for each concentration of dsRNA.

FIG. 4. Effectiveness of different transfection promoting agents on theoral delivery of dsRNA to neonate D. melanogaster larvae. A total of 20larvae were soaked in different transfection promoting agents containing1 ug/ul dsRNA, and the GUS activity was assessed in 2^(nd) instarlarvae.

FIG. 5. Gene silencing in D. melanogaster larvae after feeding neonatesdsRNA, without the presence of spermidine in the RNA mixture. Neonatelarvae were soaked in a composition comprising transfection promotingagent and GUS dsRNA, and individuals were assayed as 2^(nd) instarlarvae. A total of 25 individuals were assayed, with each dotrepresenting one individual's level of GUS gene silencing, relative tonon-treated controls.

FIG. 6. GUS gene silencing in D. melanogaster fed RNA extracts derivedfrom D. melanogaster adults that had been injected with the dsRNAexpression plasmid phspGUS[i/r] as embryos. The top panel illustratesthe range of gene silencing in 3^(rd) instar larvae previously fed theextracted RNA, and the bottom panel shows the range of gene silencingobserved in adult flies. Each dot represents a single individual insect.A total of 20 individuals were assayed for each group.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques

Unless otherwise indicated, the recombinant DNA techniques utilized inthe present invention are standard procedures, well known to thoseskilled in the art. Such techniques are described and explainedthroughout the literature in sources such as, J. Perbal, A PracticalGuide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourLaboratory Press (1989), T. A. Brown (editor), Essential MolecularBiology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M.Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach,Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al.(Editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent) and are incorporated herein by reference.

Standard methods for the production of transgenic insects are outlinedin “Insect Transgenesis—Methods and Applications” (Ed. A. M. Handler andA. A. James, CRC Press, London, 2000).

dsRNA

As used herein, “dsRNA” or “RNAi” refers to a polyribonucleotidestructure formed either by a single self-complementary RNA strand or atleast by two complementary RNA strands. The degree of complementary, inother words the % identity, need not necessarily be 100%. Rather, itmust be sufficient to allow the formation of a double-stranded structureunder the conditions employed.

Preferably, the % identity of a polyribonucleotide is determined by GAP(Needleman and Wunsch, 1970) analysis (GCG program) using the defaultsettings, wherein the query sequence is at least about 21 to about 23nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least about 21 nucleotides. In another embodiment,the query sequence is at least 150 nucleotides in length, and the GAPanalysis aligns the two sequences over a region of at least 150nucleotides. In a further embodiment, the query sequence is at least 300nucleotides in length and the GAP analysis aligns the two sequences overa region of at least 300 nucleotides. In yet another embodiment, thequery sequence corresponds to the full length of the target RNA and theGAP analysis aligns the two sequences over the full length of the targetRNA.

The design and production of suitable dsRNA molecules for the presentinvention is well within the capacity of a person skilled in the art,particularly considering Dougherty and Parks (1995), Waterhouse et al.(1998). Elbashir et al. (2001), WO 99/32619, WO 99/53050 and WO99/49029.

Conveniently, the dsRNA can be produced from a single open reading framein a recombinant host cell, wherein the sense and anti-sense sequencesare flanked by an unrelated sequence which enables the sense andanti-sense sequences to hybridize to form the dsRNA molecule with theunrelated sequence forming a loop structure.

The two strands can also be expressed separately as two transcripts, oneencoding the sense strand and one encoding the antisense strand.

RNA duplex formation can be initiated either inside or outside the cell.The dsRNA can be partially or fully double-stranded. The RNA can beenzymatically or chemically synthesized, either in vitro or in vivo.

The dsRNA need not be full length relative to either the primarytranscription product or fully processed RNA. Generally, higher identitycan be used to compensate for the use of a shorter sequence.Furthermore, the dsRNA can comprise single stranded regions as well,e.g., the dsRNA can be partially or fully double stranded. The doublestranded region of the dsRNA can have a length of at least about 21 toabout 23 base pairs, optionally a sequence of about 21 to about 50 basepairs, optionally a sequence of about 50 to about 100 base pairs,optionally a sequence of about 100 to about 200 base pairs, optionally asequence of about 200 to about 500, and optionally a sequence of about500 to about 1000 or more base pairs, up to molecule that is doublestranded for its full length, corresponding in size to a full lengthtarget RNA molecule.

The dsRNA can contain known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiralmethyl phosphonates and 2-O-methyl ribonucleotides.

As used herein, the term “specifically reduce the level of a target RNAand/or the production of a target protein encoded by the RNA”, andvariations thereof, refers to the sequence of a portion of one strand ofthe dsRNA being sufficiently identical to the target RNA such that thepresence of the dsRNA in a cell reduces the steady state level and/orthe production of said RNA. In many instances, the target RNA will bemRNA, and the presence of the dsRNA in a cell producing the mRNA willresult in a reduction in the production of said protein. Preferably,this accumulation or production is reduced at least 10%, more preferablyat least 50%, even more preferably at least 75%, yet even morepreferably at least 95% and most preferably 100%, when compared to awild-type cell.

The consequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism or by biochemical techniquessuch as, but not limited to, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), and other immunoassays.

Transfection Promoting Agent

Transfection promoting agents used to facilitate the uptake of nucleicacids into a living cell are well known within the art. Reagentsenhancing transfection include chemical families of the types;polycations, dendrimers, DEAE Dextran, block copolymers and cationiclipids. Preferably, the transfection-promoting agent is alipid-containing compound (or formulation), providing a positivelycharged hydrophilic region and a fatty acyl hydrophobic region enablingself-assembly in aqueous solution into vesicles generally known asmicelles or liposomes, as well as lipopolyamines.

The formulation of polynucleotides encapsulated in lipid-containingcompounds in known in the art and described in, for example, “Liposomes:from physical structure to therapeutic applications” (Ed. C. G. Knight.Elsevier Press, 1981).

As used herein;

-   1) CellFECTIN refers to a 1:1.5 (M/M) liposome formulation of the    cationic lipid N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II).    N^(III)-tetrapalmitylspermine (TM-TPS) and dioleoyl    phosphatidylethanolamine (DOPE) in membrane-filtered water;-   2) Lipofectin refers to a 1:1 (w/w) liposome formulation of the    cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium    chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE);-   3) Lipofectamine refers to a 3:1 (w/w) liposome formulation of the    polycationic lipid    2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate    (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine    (DOPE) in membrane-filtered water;-   4) DMRIE-C refers to a 1:1 (M/M) liposome formulation of the    cationic lipid DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy    ethyl ammonium bromide) and cholesterol in membrane-filtered water;-   5) DOTAP refers to cationic lipid    N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium    methyl-sulfate;-   6) CS096: K3C10TChol (a T-shape trilysine head group with a C10    aliphatic spacer attached to a cholesterol hydrophobic domain via    Tris molecule);-   7) CS102: K3C10TL3 (T-shape trilysine with a C10 aliphatic spacer    attached to three aliphatic fatty acid (C12) via Tris molecule);-   8) CS129: K3C7TS3 (T-shape trilysine with a C7 aliphatic spacer    attached to three aliphatic fatty acid (C18) via Tris molecule);-   9) CS078: K2C10TL3 (dilysine with a C10 aliphatic spacer attached to    three aliphatic fatty acid (C12) via Tris molecule);-   10) CS051: K3GTL3 (tri-Lysine with a shorter Glycine spacer to three    aliphatic fatty acid (C12) via Tris molecule);-   11) CS027: KATP3 (monolysine with a short alanine spacer to three    aliphatic fatty acid (C16) via Tris molecule);-   12) CS041: K3ATL2 (trilysine with a short alanine spacer to two    aliphatic fatty acid (C16) via Tris molecule);-   13) CS042: K3ATL3 (trilysine with a short alanine spacer to three    aliphatic fatty acid (C16) via Tris molecule);-   14) CS060: K3C6TL3 (trilysine with a C6 aliphatic spacer to three    aliphatic fatty acid (C16) via Tris molecule);-   15) CS039: K3ATM3 (trilysine with a short alanine spacer to three    aliphatic fatty acid (C16) via Tris molecule);-   16) CS015: K3ATP3 (trilysine with a short alanine spacer to three    aliphatic fatty acid (C16) via Tris molecule).

CS096, CS102, CS129, CS078, CS051, CS027, CS041, CS042, CS060, CS039 andCS015 are specific examples of transfection promoting agents suitablefor the methods and compositions of the invention, the method forsynthesizing which is detailed in WO 96/05218, U.S. Pat. Nos. 5,583,198,5,869,606 and 5,854,224 (see below) and Cameron et al. (1999).

As used in the present invention, the terms “micelle” and “liposome”mean vesicles composed of amphiphilic lipids self-assembled in aqueoussolution to form tertiary structures.

Liposomes are unilamellar or multilamellar vesicles of bilayers whichhave a membrane formed from a lipophilic material and an aqueousinterior, The aqueous portion may be organised to contain thecomposition to be delivered.

Cationic liposomes carry positive charges on their hydrophilichead-group forming liposomes that interact with the negatively chargednucleic acid molecules to form a complex. The positively chargedliposome/nucleic acid complex binds to the negatively charged cellsurface and is internalized predominantly through the endosomal pathway.A proportion of the endosomes, will rupture, releasing their contents ofliposome/nucleic acid complex into the cell cytoplasm.

Liposomes that are pH-sensitive or negatively charged, entrap nucleicacid rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, nucleic acid can be entrapped within the aqueousinterior of these liposomes.

One major type of liposomal composition includes phospholipids otherthan naturally derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, or (B) is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Whilenot wishing to be bound by any particular theory, it is thought in theart that, at least for sterically stabilized liposomes containinggangliosides, sphingomyelin, or PEG-derivatized lipids, the enhancedcirculation half-life of these sterically stabilized liposomes derivesfrom a reduced uptake into cells of the reticuloendothelial system (RES)(Allen and Chonn, 1987; Wu et al., 1993).

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 discloses methods for encapsulating high molecular weightnucleic acids in liposomes. U.S. Pat. No. 5,264,221 disclosesprotein-bonded liposomes and asserts that the contents of such liposomescan include an antisense RNA. U.S. Pat. No. 5,665,710 describes certainmethods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787discloses liposomes comprising antisense oligonucleotides targeted tothe raf gene.

Transfection promoting agents useful for the methods and compositions ofthe present invention include “Tris cationic lipids” which are disclosedin WO 96/05218, U.S. Pat. Nos. 5,854,224, 5,583,198 and 5,869,606, thecontents of which are incorporated by reference. These agents includecompounds having a formula selected from the group consisting of:

in which:

w is a dsRNA or a nucleic acid encoding a dsRNA

x is a peptide, amino acid, non-amino acid nucleic acid binding group ornon-peptide nucleic acid binding group

y is a linker having a chain length equivalent to 1 to 20 carbon atomsor is absent

R₄ is H or CH₂O—R₃; and R₁, R₂ and R₃ are the same or different and areeither hydrogen, methyl, ethyl, hydroxyl or an acyl group derived from afatty acid having a carbon chain of 3 to 24 carbon atoms saturated orunsaturated, with the proviso that at least one of R₁, R₂ and R₃ is anacyl group derived from a fatty acid;w . . . x-y-NH—CH₂—CH₂O—R₅  ii)in which:

w is a dsRNA or a nucleic acid encoding a dsRNA

x is a peptide, amino acid, non-amino acid nucleic acid binding group ornon-peptide nucleic acid binding group

y is a linker having a chain length equivalent to 1 to 20 carbon atomsor is absent

R₅ is an acyl group derived from a fatty acid having a carbon chain of 3to 24 carbon atoms saturated or unsaturated;

in which:

w is a dsRNA or a nucleic acid encoding a dsRNA

x is a peptide, amino acid, non-amino acid nucleic acid binding group ornon-peptide nucleic acid binding group

y is a linker having a chain length equivalent to 1 to 20 carbon atomsor is absent

R₄ is H or CH₂O—R₃; and R₁, R₂ and R₃ are the same or different and areeither hydrogen, methyl, ethyl, hydroxyl or an acyl group derived from afatty acid having a carbon chain of 3 to 24 carbon atoms saturated orunsaturated, with the proviso that at least one of R₁, R₂ and R₃ is anacyl group derived from a fatty acid;w . . . x-y-NH—CH₂—CH₂O—R₅  iv)in which:

w is a dsRNA or a nucleic acid encoding a dsRNA

x is a peptide, amino acid, non-amino acid nucleic acid binding group ornon-peptide nucleic acid binding group

y is a linker having a chain length equivalent to 1 to 20 carbon atomsor is absent

R₅ is an acyl group derived from a fatty acid having a carbon chain of 3to 24 carbon atoms saturated or unsaturated; and

in which:

w is a dsRNA or a nucleic acid encoding a dsRNA

x is a peptide, amino acid, non-amino acid nucleic acid binding group ornon-peptide nucleic acid binding group

y is a spacer having a chain length equivalent to 1-30 carbon-carbonsingle covalent bonds or is absent

R₄ is H or halogen or CH₂O—R₃; and R₁, R₂ and R₃ are the same ordifferent and are either hydrogen, methyl, ethyl, alkyl, alkenyl,hydroxylated alkyl, hydroxylated alkenyl groups or ether containingalkyl, alkenyl, hydroxylated alkyl or hydroxylated alkenyl groups,optionally being an acyl group derived from a fatty acid having a carbonchain length equivalent to 3-24 carbon atoms saturated or unsaturated,with the proviso that at least one of R₁, R₂ and R₃ includes a grouphaving a carbon chain of 3-24 carbon atoms saturated or unsaturated.

Within the meaning of the present invention, the term lipopolyaminedenotes any amphiphilic molecule comprising at least one hydrophilicpolyamine region and one lipophilic region. The cationically chargedpolyamine region of the lipopolyamines is capable of combiningreversibly with the negatively charged nucleic acid. This interactionstrongly compacts the nucleic acid. The lipophilic region makes thisionic interaction less sensitive to the external medium, by covering thenucleolipid particle formed with a lipid layer. Examples of suitablelipopolyamines include those disclosed in U.S. Pat. Nos. 6,172,048 and6,171,612.

Advantageously, the polyamine region of the lipopolyamines used in thecontext of the invention corresponds to the general formula:H₂N—(—(CH)_(m)—NH—)_(n)—Hin which m is an integer greater than or equal to 2 and n is an integergreater than or equal to 1, it being possible for m to vary between thedifferent carbon groups included between two amines. Preferably, m isbetween 2 and 6 inclusive and n is between 1 and 5 inclusive. Still morepreferably, the polyamine region is represented by spermine or ananalogue of spermine that has retained its properties of binding tonucleic acids.

The lipophilic region can be a saturated or unsaturated hydrocarbonchain, cholesterol, a natural lipid or a synthetic lipid capable offorming lamellar, cubic, or hexagonal phases.

There was some variation in effectiveness of the transfection reagentstested in the arthropod species that were examined. However, consideringthe present disclosure, it is well within the capacity of the skilledaddressee to design routine experiments to test a number of transfectionpromoting agents to determine which provides the best results for anygiven arthropod species.

Agriculturally Acceptable Carriers

Agriculturally suitable and/or environmentally acceptable compositionsfor arthropod control are known in the art. Agricultural compositionsfor the control of arthropod pests of plants and/or animals arepreferably suitable for agricultural use and dispersal in fields.Preferably, compositions for the control of other arthropod pests shouldbe environmentally acceptable.

Agriculturally acceptable carriers are also referred to herein as an“excipient”. An excipient can be any material that the animal, plant orenvironment to be treated can tolerate. Furthermore, the excipient mustbe such that the composition of the present invention is still capableof causing gene silencing. Examples of such excipients include water,saline. Ringers solution, dextrose or other sugar solutions, Hank'ssolution, and other aqueous physiologically balanced salt solutions,phosphate buffer, bicarbonate buffer and Tris buffer. In addition, thecomposition may include compounds that increase the half-life of acomposition. Such compounds are be known to the skilled person in theart.

Compositions of the invention may also comprise agents selected from;conventional pesticides, gustatory stimulants, thickening agents, UVscreening agents, optical brighteners, dispersants, flow agents,spreading agents and sticking agents. Preferably, the composition isformulated such that is persist in the environment for a length of timesuitable to allow it to be ingested by a target arthropod or contact thetarget arthropod.

Arthropods

The arthropod can be any organism classified in this taxonomical group.Preferably, the arthropod is selected from the group consisting of:Crustacea. Insecta and Arachnida.

Examples of preferred Insecta include, but are not limited to, membersof the orders Coleoptera (e.g. Anobium, Ceutorhynchus, Rhynchophorus,Cospopolites, Lissorhoptrus, Meligethes, Hypothenemus, Hylesinus,Acalymma, Lema, Psylliodes, Leptinotarsa, Gonocephalum, Agriotes,Dermolepida, Heteronychus, Phaedon, Tribolium, Sitophilus, Diabrotica,Anthonomus or Anthrenus spp.), Lepidoptera (e.g. Ephestia, Mamestra,Earias, Pectinophora, Ostrinia, Trichoplusia, Laphygma, Agrotis,Amathes, Wiseana, Tryporyza, Diatraea, Sporganothis, Cydia, Archips,Plutella, Chilo, Heliothis, Helicoverpa (especially Helicoverpaarmigera), Spodoptera or Tineola ssp.), Diptera (e.g. Musca, Aedes,Anopheles, Culex, Glossina, Simulium, Stomoxys, Haematobia, Tabanus,Hydrotaea, Lucilia, Chrysomia, Callitroga, Dermatobia, Gasterophilus,Hypoderma, Hylemyia, Atherigona, Chlorops, Phytomyza, Ceratitis,Liriomyza, and Melophagus spp.), Phthiraptera, Hemiptera (e.g. Aphis,Bemisia, Phorodon, Aeneoplamia, Empoasca, Parkinsiella, Pyrilla,Aonidiella, Coccus, Pseudococcus, Helopeltis, Lygus, Dysdercus,Oxycarenus, Nezara, Aleurodes, Triatoma, Rhodnius, Psylla, Myzus,Megoura, Phylloxera, Adelyes, Niloparvata, Nephrotettix or Cimex spp.),Orthoptera (e.g. Locusta, Gryllus, Schistocerca or Acheta spp.),Dictyoptera (e.g. Blattella, Periplaneta or Blatta spp.), Hymenoptera(e.g. Athalia, Cephus, Atta, Lasius, Solenopsis or Monomorium spp.),Isoptera (e.g. Odontotermes and Reticulitermes spp.), Siphonaptera (e.g.Ctenocephalides or Pulex app). Thysanura (e.g. Lepisma spp.), Dermaptera(e.g. Forficula spp.) and Psocoptera (e.g. Peripsocus spp.) andThysanoptera (e.g. Thrips tabaci). In one embodiment, the Arthropod isnot a Drosophila sp.

Examples of preferred Arachnida include, but are not limited to, ticks,e.g. members of the genera Boophilus, Ornithodorus, Rhipicephalus,Amblyomma, Hyalomma, Ixodes, Haemaphysalis, Dermocentor and Anocentor,and mites and manges such as Acarus, Tetranychus, Psoroptes, Notoednes,Sarcoptes, Psorergates, Chorioptes, Demodex, Panonychus, Bryobia andEriophyes spp.

Examples of preferred Crustaceans include, but are not limited to,crayfish, prawns, shrimps, lobsters and crabs.

Recombinant Vectors

Polynucleotides encoding dsRNA useful for the methods and/orcompositions of the present invention can be inserted into a recombinantvector. The vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide encoding adsRNA operatively linked to an expression vector. Alternatively, the twostrands of the dsRNA are encoded by separate open reading frames. Thephrase operatively linked refers to insertion of a polynucleotidemolecule into an expression vector in a manner such that the molecule isable to be expressed when transformed into a host cell. As used herein,an expression vector is a DNA or RNA vector that is capable oftransforming a host cell and of effecting expression of a specifiedpolynucleotide molecule(s). Preferably, the expression vector is alsocapable of replicating within the host cell. Expression vectors can beeither prokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, endoparasite,arthropod, other animal, and plant cells. Preferred expression vectorsof the present invention can direct gene expression in arthropod cells.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences, origins ofreplication, and other regulatory sequences that are compatible with therecombinant cell and that control the expression of the polynucleotideencoding a dsRNA or a strand thereof. In particular, recombinantmolecules of the present invention include transcription controlsequences. Transcription control sequences are sequences which controlthe initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation, such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in at leastone of the recombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin arthropod cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers.

A particularly preferred expression vector is a baculovirus. By“baculovirus” it is meant any virus of the family Baculoviridae, such asa nuclear polyhedrosis virus (NPV). Baculoviruses are a large group ofevolutionarily related viruses, which infect only arthropods; indeed,some baculoviruses only infect insects that are pests of commerciallyimportant agricultural and forestry crops, while others are known thatspecifically infect other insect pests. Because baculoviruses infectonly arthropods, they pose little or no risk to humans, plants, or theenvironment.

Of the suitable DNA viruses, in addition to the Baculoviridae are theentomopox viruses (EPV), such as Melolontha melonotha EPV. Amsactamoorei EPV. Locusta migratoria EPV. Melanoplus sanguinipes EPV.Schistocerca gregaria EPV, Aedes aogypti EPV, and Chironomus luridusEPV. Other suitable DNA viruses are granulosis viruses (GV). SuitableRNA viruses include togaviruses, flaviviruses, picornaviruses,cytoplasmic polyhedrosis viruses (CPV), and the like. The subfamily ofdouble stranded DNA viruses Eubaculovirinae includes two genera, NPVsand GVs, which are particularly useful for biological control becausethey produce occlusion bodies in their life cycle. Examples of GVsinclude Cydia pomonella GV (coddling moth GV). Pieris brassicae GV.Trichoplusia ni GV. Artogeia rapae GV, and Plodia interpunctella GV(Indian meal moth).

Suitable baculoviruses for practicing this invention may be occluded ornon-occluded. The nuclear polyhedrosis viruses (“NPV”) are onebaculovirus sub-group, which are “occluded.” That is, a characteristicfeature of the NPV group is that many virions are embedded in acrystalline protein matrix referred to as an “occlusion body.” Examplesof NPVs include Lymantria dispar NPV (gypsy moth NPV), Autographacalifornica MNPV, Anagrapha falcifera NPV (celery looper NPV),Spodoptera litturalis NPV, Spodoptera frugiperda NPV, Heliothis armigeraNPV, Mamestra brassicae NPV, Choristoneura fumiferana NPV, Trichoplusiani NPV, Helicoverpa zea NPV, and Rachiplusia ou NPV. For field useoccluded viruses often are preferable due to their greater stabilitysince the viral polyhedrin coat provides protection for the enclosedinfectious nucleocapsids.

Among illustrative, useful baculoviruses in practicing this inventionare those isolated from Anagrapha falcifera, Anticarsia gemmatalis,Buzura suppressuria, Cydia pomonella, Helicoverpa zea, Heliothisarmigera, Manestia brassicae, Plutella xylostella, Spodoptera exigua,Spodoptera littoralis, and Spodoptera litura. A particularly useful“NPV” baculovirus for practicing this invention is AcNPV, which is anuclear polyhedrosis virus from Autographa californica. Autographacalifornica is of particular interest because various major pest specieswithin the genera Spodoptera, Trichoplusia, and Heliothis aresusceptible to this virus.

Transgenic Plants

The term “plant” refers to whole plants, plant organs (e.g. leaves,stems roots, etc), seeds, plant cells and the like. Plants contemplatedfor use in the practice of the present invention include bothmonocotyledonous and dicotyledonous plants. Exemplary dicotyledonousplants include cotton, oilseeds and other brassicas, tomato, tobacco,potato, bean, and soybean. Exemplary monocotyledonous plants includewheat, maize, barley, rice, and sorghum. The choice of the plants peciesis determined by the intended use of the plant or parts thereof and theamenability of the plant species to transformation.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinant DNAtechniques to produce at least one dsRNA useful for the methods of thepresent invention in the desired plant or plant organ.

A polynucleotide encoding a dsRNA, or two different polynucleotidesencoding individual strands of a dsRNA, may be expressed constitutivelyin the transgenic plants during all stages of development. Depending onthe use of the plant or plant organs, the dsRNA may be produced in astage-specific manner. Furthermore, depending on the use, thepolynucleotides may be expressed tissue-specifically or induced underspecific environmental condition such as for example, wounding by anarthropod pest.

Regulatory sequences which are known or are found to cause expression ofa polynucleotide(s) encoding a dsRNA of interest in plants may be usedin the present invention. The choice of the regulatory sequences useddepends on the target crop and/or target organ of interest and thedesired mode of expression (e.g. constitutive induced or tissuespecific). Such regulatory sequences may be obtained from plants orplant viruses, or may be chemically synthesized. Such regulatorysequences are well known to those skilled in the art.

Other regulatory sequences such as terminator sequences andpolyadenylation signals include any such sequence functioning as such inplants, the choice of which are known to the skilled addressee. Anexample of such sequences is the 3′ flanking region of the nopalinesynthase (nos) gene of Agrobacterium tumefaciens.

Several techniques are available for the introduction of an expressionconstruct containing a polynucleotide(s) encoding a dsRNA of interestinto the target plants. Such techniques include but are not limited totransformation of protoplasts using the calcium/polyethylene glycolmethod, electroporation and microinjection or (coated) particlebombardment. In addition to these so-called direct DNA transformationmethods, transformation systems involving vectors are widely available,such as viral and bacterial vectors (e.g. from the genus Agrobacterium).After selection and/or screening, the protoplasts, cells or plant partsthat have been transformed can be regenerated into whole plants, usingmethods known in the art. The choice of the transformation and/orregeneration techniques is not critical for this invention.

EXAMPLES

Methods

GUS RNA In Vitro Transcription Plasmids

Standard gene cloning methods (Sambrook et al., 1989) were used to makethe gene constructs. The GUS gene encoding the bacterial enzymeβ-glucuronidase was amplified by PCR from the pBacPAK8-GUS plasmid(Clonetech) using the primers EcoGusF (GAATTCATGGTCCGTCCTGTAGAAACC) (SEQID NO: 1) and EcoGusR (GAATTCCCCCACCGAGGCTGTAGC) (SEQ ID NO: 2). The1.87 kb PCR product was subcloned into the plasmid pGEM3Zf(+) into theEcoR I site using the EcoR I linkers on the primers, creating twoplasmids: pGEM3Z-GUS[s] (sense orientation of GUS gene, relative to theT7 promoter); and pGEM3Z-GUS[a/s] (antisense orientation of GUS gene,relative to the T7 promoter). Both plasmids were digested with therestriction endonuclease EcoRV, followed by relegation of the plasmid,to remove 213 bp of the GUS ORF. This ensured that no functional GUSenzyme would be produced if the sense GUS RNA was translated. Theresultant plasmids, named pGEM3Z-ΔGUS[s] and pGEM3Z-ΔGUS[a/s], were usedfor in vitro transcription of sense and antisense GUS RNAs.

GUS RNA In Vivo Expression Constructs

In vivo expression of sense, antisense, and inverted repeat RNA in D.melanogaster embryos was achieved by preparing three plasmids thatexpressed RNA under the control of the D. melanogaster heat shockpromoter hsp70. A 1 kb fragment containing the hsp70 promoter, a smallmultiple cloning site, and the heat shock polyadenylation signal wasamplified using PCR from the plasmid pCaSpeR-hs (Thummel et al., 1988)using the primers hsp70F (GAATTCTAGAATCCCAAAACAAACTGG) (SEQ ID NO: 3)and hst70R (GGATCCTGACCGTCCATCGCAATAAAATGAGCC) (SEQ ID NO: 4).

The 1 kb PCR product was cloned into pGEM-T-Easy, resulting in theplasmid pGEM-Dmhsp70. The GUS gene was excised from the plasmidpGEM3Z-GUS[s] using the restriction endonuclease EcoRI, and ligated intothe pGEM-Dmhsp70 plasmid, previously linearized with EcoRI. Thisligation resulted in two plasmids, phspGUS[s], with the GUS gene in thesense orientation with respect to the promoter, and phspGUS[a/s], withthe GUS gene in the antisense orientation. A third plasmid,pHSP70GUS[i/r], was prepared that expressed an inverted repeat dsRNAspecific to the GUS open reading frame (ORF), by ligating a 558 bp DNAfragment, representing the 5′ end of the GUS gene, to the 3′ end of theGUS ORF. The resulting coding sequence, when transcribed, could producea transcript with complementary sequences at the 5′ and 3′ ends, whichcould fold back upon itself to form a hairpin dsRNA, withdouble-stranded sequence for 558 bases.

H. armigera vATPase In Vitro Transcription Plasmids

A 386 bp segment of a putative vATPase gene was amplified from H.armigera genomic DNA using the two primers HaATP1f(CCGAAAATCCAATCTACGGACCC) (SEQ ID NO: 5) and HaATP1r(CGACGAATAACCTGGGCTGTTGC) (SEQ ID NO: 6). The primers were based on DNAsequence of a putative vATPase gene identified from a H. armigera ESTclone that showed 97% sequence identity to the vATPase gene of Heliothisvirescens (GenBank accession # L16884). The 386 bp product was amplifiedusing a Perkin Elmer 2400 Thermocycler using the following PCRconditions: 1 cycle of 95° C. for 5 min, 25 cycles of 95° C.×30 sec, 55°C.×30 sec, 72° C.×30 sec, and 1 cycle of 72° C.×10 min, 25° C.×5 min.The PCR product was ligated into the pGem-T-Easy cloning vector(Promega) in both orientations with respect to the T7 promoter,producing the plasmids pGEMHaATPase1[s] and pGEMHaATPase1[a/s], whichwere used to produce in vitro transcribed sense and antisense vATPaseRNAs.

Drosophila Transformation

The GUS gene encoding the bacterial enzyme β-glucuronidase, was insertedinto the P-element transformation vector pCaSpeR-act, which placed theGUS gene under the control of the act5c promoter. The GUS gene was thenintroduced into the Drosophila germline by P-element transformation(Spradling and Rubin, 1982). Transformants were backcrossed tochromosomal balancer strains to identify into which chromosome thetransgene had inserted. Southern analyses of DNA from G2 flies werepreformed to determine the copy number of the transgene in the GUStransgenic stock.

Preparation of Double-Stranded RNA by In Vitro Transcription

The plasmids pGEM3Z-ΔGUS[s] and -ΔGUS [a/s] were linearized using BamHI. Sense and antisense RNA was prepared using T7 RNA polymerase usingPromega's RiboMAX Large Scale RNA Production System, according to themanufacturer's instructions. To produce dsRNA, sense and antisense RNAswere mixed in equimolar quantities and annealed for 10 minutes at 37° C.The RNA was extracted with phenol/chloroform and then chloroform,precipitated with ethanol, and resuspended in 10 mM Tris-HCl, pH 9.Formation of dsRNA was confirmed by resolving the annealed andnon-annealed RNAs on a 1.0% agarose gel in TBE (90 mM Tris-borate, 2 mMEDTA, pH 8.0).

To produce vATPase dsRNA, the plasmids pGEMHaATPase1[s] andpGEMHaATPase1[a/s] were linearized with Bam HI and sense, antisense, anddouble-stranded RNAs were produced as described above.

Embryo Injections

Preblastoderm D. melanogaster embryos were microinjected with DNA or RNAaccording to the method of Spradling and Rubin (1982) and H. armigeraembryos were microinjected as previously described (Pinkerton et al.,1996). The embryos were injected with sense, antisense, and dsRNAsdissolved in injection buffer (5 mM KCl, 0.1 mM PO₄, pH 6.8) at aconcentration of 100 ng/ul. Approximately 50 pg of RNA were injected ineach embryo. Negative control embryos were mock-injected with injectionbuffer alone. Embryos injected with DNA were injected with approximately250 pg of plasmid DNA. The embryos were permitted to fully develop for16 h, and were either snap frozen for use in subsequent GUS assays orwere permitted to hatch and surviving larvae were transferred to vialscontaining culture medium. Individual larvae and adult insects werecollected and snap frozen at −80° C.

Oral dsRNA Delivery

Newly hatched 1^(st) instar larvae (Drosophila melanogaster orHelicoverpa armigera) were transferred to 96-well plates in groups of10-25, and washed in phosphate buffered saline (PBS). Sense, antisense,and annealed dsRNAs (0.05-2 ug) were mixed with 1 ul of transfectionpromoting agent, 0.5 mM spermidine or protamine sulphate (0.5 mg/mgDNA), in a volume of 20 ul of PBS or buffered sucrose (20% sucrose, 10mM Tris, pH 7.5). After 30 min, red food dye was added to thetransfection promoting agent-RNA mixture and the mixture was added tothe neonate larvae. The larvae remained immersed in the mixtures for 1h, and larvae were then transferred to rearing medium. Approximately 90%of individuals treated in this manner contained red food dye in theirguts, indicating that most had ingested the mixture.

Rearing Conditions

D. melanogaster were raised at 25° C. on standard yeast-agar Drosophilaculture media (Roberts and Standen, 1998). H. armigera were raised aspreviously described (Duve et al., 1997).

GUS Assays

Insects were homogenised in homogenisation buffer (50 mM NaHPO₄, pH 7.0,10 mM p-mercaptoethanol, 10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1%Triton X-100), and GUS enzyme activity was measured using4-methylumbelliferyl p-D-glucuronic acid as a substrate in fluorometricassays as described (Gallagher, 1992). Protein assays were performedusing the Bradford assay (Bradford, 1976). Dissected insects werestained for GUS activity using 5-bromo-4-chloro-3-indolyl p-D-glucuronicacid (X-GlcU) as described (Naleway, 1992).

Results

Characterisation of the GUS Transgenic Strain

Standard genetic and Southern analyses confirmed that the GUS transgenicstock of D. melanogaster contained a single insertion of the act5c-GUSconstruct, located on chromosome III (results not shown). The GUS genewas constitutively expressed throughout the body, with extensive GUSactivity observed in the fat body and gonads of both males and females(data not shown). Fluorometric GUS enzyme assays confirmed that alldevelopmental stages of the GUS transgenics had at least 18 times moreGUS activity than their non-transformed counterparts (Table 1).

Silencing of the GUS Gene in Drosophila Embryos Using In VitroTranscribed and Annealed Double Stranded RNA

Following injection of RNA into preblastoderm embryos, embryos werepermitted to develop for 16 h, just prior to hatching, before they wereassayed for GUS activity. Embryos were pooled in groups of 25, whereaslarvae and adults were assayed individually for GUS activity. While itwas not possible to determine which individual embryos were mostaffected by the RNA injections, it was clear that both sense andantisense had no or little affect on GUS activity, whereas those embryosinjected with dsRNA showed significant reductions in GUS activity (Table2). Northern analyses of RNA from mock-injected and dsRNA-injectedembryos confirmed that the reduction of GUS activity correlated withreduction of GUS transcripts in the dsRNA-injected embryos (results notshown). Interestingly, the silencing of the GUS gene expressionpersisted throughout development, as both larvae and adults that hadbeen treated with dsRNA as embryos still showed substantial reductionsin GUS activity. These results confirmed that GUS gene expression couldbe effectively reduced by direct delivery of in vitro-prepared dsRNAinto the embryos.

TABLE 1 GUS activity in non-transgenic and transgenic D. melanogasterFold increase in Developmental GUS activity (pmol MU/min/individual) GUSactivity in stage Non-transgenic Transgenic transgenics embryo  74 ± 20 1980 ± 152 27 3^(rd) instar larva 417 ± 34  7390 ± 780 18 adult 574 ±55 12620 ± 827 22

TABLE 2 Reduction of GUS activity following embryonic injection of RNAto D. melanogaster GUS strain embryos. Values represent the percentagedecrease (±standard error) of GUS activity relative to mock-injectedembryos. Reduction in GUS activity following delivery of RNA (%)Embryos¹ Larvae² Adults² Sense RNA 2 ± 1 3 ± 2  5 ± 3 Antisense RNA 9 ±4 7 ± 5 15 ± 8 ds RNA 65 ± 14 41 ± 7  32 ± 5 ¹Values represent resultsfrom 3 separate replicates of 25 embryos each. ²Values represent resultsfrom 3 separate replicates of 10 individuals each.Silencing of the GUS Gene in Drosophila Embryos Using In Vivo-ProduceddsRNA

GUS strain embryos were injected with the plasmids phspGUS[s],phspGUS[a/s], and phspGUS[i/r], and then heat shocked 6 h postinjection. The embryos were collected just prior to hatching (16 hdevelopment), and were assayed for GUS activity. The embryos injectedwith phspGUS[s] showed no difference in GUS activity, whereas embryosinjected with phspGUS[a/s] showed a 12% decrease in GUS activityrelative to mock-injected controls (Table 3). Embryos injected with theinverted repeat RNA expression construct, phspGUS[i/r], showedsubstantial (90%) reduction of GUS activity. Adults that developed fromembryos injected with the phspGUS[i/r] plasmid showed persistence of thegene silencing phenotype, having a 55% reduction in GUS activityrelative to mock-injected controls. Adults derived from injections ofplasmids that expressed sense or antisense RNA showed no persistence ofthe gene silencing.

TABLE 3 Reduction of GUS activity following embryonic injection of RNA-expression plasmids to D. melanogaster GUS strain embryos. Valuesrepresent the percentage decrease (±standard error) of GUS activityrelative to mock-injected embryos. Reduction in GUS activity(%) EmbryosAdults phspGUS[s]  1 ± 1  1 ± 2 phspGUS[a/s] 12 ± 2  2 ± 1 hspGUS[i/r]90 ± 8 55 ± 6

PCR analysis of different developmental stages showed that the injectedplasmid could not be detected beyond first instar larvae (FIG. 1),suggesting that the injected DNA was quickly degraded once the insectsmolted into 2^(nd) instar larvae. The persistence of the gene silencingthroughout development was therefore most likely due to the persistenceof the dsRNA, and not due to sustained expression of dsRNA from theinjected plasmid.

Silencing of the GUS Gene in Drosophila Following Soaking of Larvae indsRNA

Drosophila larvae fed naked GUS dsRNA showed no changes in GUS geneexpression (results not shown). Similarly, no change in GUS activity in2nd instar larvae or adults was observed when neonate larvae wereimmersed in a DMRIE-C mixture containing GUS sense or antisense RNA(results not shown). In contrast, 15% of neonates soaked in transfectionpromoting agent containing GUS dsRNA developed into adult flies thatshowed >90% reduction of GUS activity (FIG. 2). Another 35% of thesurviving flies showed an intermediate (20-80%) reduction of GUSexpression. Similarly, 2^(nd) instar larvae derived from neonates soakedin dsRNA showed a similar result, with 20% of larvae having >90%reduction of GUS activity, and another 40% of the larvae showing areduction of GUS activity between 20% and 80% of normal GUS activitylevels. These results indicate that in vitro transcribed and annealeddsRNA can be fed to neonates and cause extensive, body-wide genesilencing of the target gene. This method of dsRNA delivery seemsrelatively benign, as no larvae were observed to die or suffer from thetransfection promoting agent treatment. Gene silencing appears to begene specific, as the insects showing reduced GUS activity appearedhealthy and showed no other observable phenotype.

As the larvae were soaking in the mixture, it is possible that entry ofdsRNA may have occurred either by ingestion, perfusion into the trachea,or by absorption through the cuticle. However, a small percentage (10%)of surviving larvae were observed not to have any food colouring intheir guts. These individuals showed no reduction of GUS activity, whichsuggested that the primary route of entry for the dsRNA is via thealimentary canal (results not shown).

The concentration of dsRNA fed to the larvae correlated directly withthe number of individuals that exhibited strong suppression of GUSactivity. The lowest concentration (0.25 ug/u|) of dsRNA tested, usingDMRIE-C, produced 4/20 flies that displayed a reduction of GUS activitygreater than 25% (FIG. 3). In contrast, the highest concentration ofdsRNA tested (1.0 ug/ul) produced 12/20 flies with a reduction of GUSactivity greater than 25%. At this highest dose, the greatest number offlies (5/20) showed a reduction of GUS activity of greater than 80%.While these sample sizes are small (20 individuals/treatment), theyindicate that the extent of gene silencing may be dsRNA dose-dependent.

Lipofectamine. Cellfectin, and DMRIE-C (Life Technologies), eachproduced individuals with a measurable level of reduced GUS activity(FIG. 4). DMRIE-C provided the greatest number of individuals withextensive gene silencing, with 25% of the larvae having greater than 75%of the GUS activity eliminated. Two individuals out of 20 showed 100%gene silencing using this transfection promoting agent. Transfectionswith Lipofectamine and Cellfectin resulted in 26-50% silencing of theGUS gene in 35% of the larvae tested, which indicates that thesetransfection promoting agents could also serve to deliver dsRNA toDrosophila via ingestion.

Given that most (approximately 70%, results not shown) of the GUS geneexpression is found in the fat body and gonads, the silencing signal hadobviously passed beyond the gut tissues and spread throughout the body.This gene silencing spreading phenomenon is not unlike that seen in C.elegans in nematodes fed dsRNA. However, it is surprising to observegene silencing in the insect following this mode of delivery of dsRNA,as the gut of Drosophila is physically and physiologically more complexthan that of C. elegans. Most notably, Drosophila produces a peritrophicmembrane throughout the length of the midgut, which theoretically couldpotentially reduce or prevent transmission of dsRNA to the midgut cells.

Addition of a nucleic acid condensing agent (spermidine or protaminesulfate) to the RNA mixture was found to enhance the efficiency of RNAiin Drosophila. Without adding spermidine, only 20% of treated larvaedisplayed a reduction of GUS activity greater than 20%, and only amaximum of 32% GUS silencing was observed (FIG. 5). Not only did thepercentage of individuals with significant levels of GUS gene silencingincrease by using spermidine, but the maximum level of GUS genesilencing increased to 100% in some individuals (see FIG. 2). A similarenhancement of RNAi was observed if protamine sulphate was used insteadof spermidine (results not shown).

The efficiency of RNAi in Drosophila was found to improve slightly whenPBS was replaced with buffered sucrose during the mixing of the RNA withthe transfection promoting agents (Table 4). Although it has not beenexamined further, it is anticipated that replacement of PBS with sucrosewill improve efficiency of the packaging of the RNA in many of thetransfection promoting agents under consideration.

A selection of transfection promoting agents was kindly provided byTrevor Lockett and colleagues (CSIRO Molecular Science). Thesetransfection promoting agents are thoroughly described in the patent“Delivery of Nucleic Acids” (PCT/AU95/00505, U.S. Pat. No. 5,906,922). Acomparison of 11 of these CSIRO reagents with the 5 commerciallyavailable reagents was conducted, and many of the CSIRO liposomes weremore effective at producing an RNAi effect in Drosophila (Table 5). Inparticular, liposomes CS096, CS102, and CS129 performed better than thebest-performing commercially available liposome, DMRIE-C. All of theCSIRO liposomes tested produced a greater number of individuals affectedby RNAi than the poorest commercially available liposome, DOTAP. Theseresults confirm that optimised delivery of dsRNA to insects can beachieved by selecting appropriate transfection promoting agents.

TABLE 4 Percentage of 2^(nd) instar larvae showing greater than a 25%reduction of GUS gene activity following soaking in transfectionpromoting agents that were mixed with RNA in either PBS or bufferedsucrose solutions. Values represent the mean and standard deviation fromtwo replicates of 25 insects. Transfection Promoting Agent PBS BufferedSucrose DMRIE-C 60 ± 7 72 ± 10 Lipofectamine 35 ± 6 49 ± 8  DOTAP  0 ± 05 ± 3Silencing of an Endogenous Gene in H. armigera

Neonate H. armigera were soaked in a composition containing transfectionpromoting agent and dsRNA specific to a putative vacuolar ATPase gene.Several vATPase genes are present in Lepidoptera, some of which areknown to encode subunits of proton pumps in the midgut cells. Theseproton pumps are responsible for establishing and maintaining the highpH (approximately pH 10) environment of the lepidopteran midgut Whileall Drosophila larvae survived the soaking treatment, only 64% of H.armigera larvae were alive 24 h after exposure to transfection promotingagent containing no RNA (Table 6). A similar percentage of caterpillars(62%) survived a treatment containing transfection promoting agent mixedwith GUS dsRNA. Only 40% of larvae soaked in transfection promotingagent mixed with vATPase dsRNA survived the first 24 h. In addition to aslightly reduced survival after the first 24 hours, delayed developmentwas also observed for larvae exposed to vATPase dsRNA.

Of those larvae surviving beyond 24 h, 85% of the control larvae reachedpupation by day 10. In contrast, only 40% of surviving larvae treatedwith vATPase dsRNA pupated by day 10. The overall mortality for larvaetreated with vATPase dsRNA, relative to those treated with transfectionpromoting agent alone was 52%. Larvae treated with GUS dsRNA were notsignificantly affected, as 82% had pupated by day 10. Oral delivery ofvATPase dsRNA therefore resulted in both reduced survival and delayeddevelopment in H. armigera larvae.

TABLE 5 Ordered ranking of CSIRO transfection promoting agents andcommercially available transfection promoting agents in their ability toinduce RNAi of the GUS transgene in Drosophila. The percentage of 2^(nd)instar larvae having greater than a 25% reduction in GUS activity wasdetermined after neonate larvae were soaked in the transfectionpromoting agent containing GUS dsRNA in buffered sucrose. Valuesrepresent the mean and standard deviation from two experiments with 15insects each. Transfection Promoting % 2^(nd) instar larvae with RankingAgent >25% GUS RNAi 1 CS096 70 ± 5 2 CS102  63 ± 14 2 CS129 63 ± 5 3DMRIE-C  56 ± 14 4 CS078 46 ± 9 5 CS051  43 ± 14 5 CS027 43 ± 5 6 CS04140 ± 9 7 Lipofectamine 36 ± 5 8 CS042 23 ± 5 9 Cellfectin 20 ± 9 9 CS06020 ± 9 10 Lipofectin 16 ± 5 11 CS039 10 ± 5 11 CS015 10 ± 5 12 DOTAP  3± 5 * Complete names of the transfection promoting agents are providedin the “Transfection Promoting Agent” section of the DetailedDescription. Lipofectin, Lipofectamine, Cellfectin, and DMRIE-C wereobtained from Life Technologies, whereas DOTAP was obtained fromBoehringer Mannheim.

Little is known about the expression of the particular vATPase gene thatwas targeted, other than that it is expressed in gut tissues (as it wasisolated from a gut-specific EST library). It is not presently known ifthe targeted vATPase gene is also expressed elsewhere in the body, norif the extent of gene silencing was sufficient to reduce the majority ofvATPase activity. Nevertheless, the GUS dsRNA produced no deleteriouseffect on the caterpillars, which indicates that the vATPasedsRNA-mediated gene silencing was sufficiently effective to cause asignificant level of mortality and morbidity.

Unlike D. melanogaster, the use of Lipofectamine provided the best RNAi(Table 7). As with Drosophila, treatments of RNA alone or RNA withspermidine failed to result in observable RNAi.

TABLE 6 Effects of soaking H. armigera larvae in transfection promotingagent containing dsRNA. The results represent the mean and standarderrors for three separate experiments using 20 larvae for eachtreatment. % surviving % pupated by % survival to larvae at 24 h ^(a)day 10 ^(b) adulthood ^(c) Transfection Promoting 64 ± 5 85 ± 5 100Agent alone Transfection Promoting 60 ± 6 82 ± 6 91 Agent + GUS dsRNATransfection Promoting  40 ± 12 40 ± 5 52 Agent + vATPase dsRNA ^(a)based on three experiments using 20 insects each ^(b) percentage basedon those insects surviving past 24 h post treatment ^(c) percentagesurvival relative to the transfection promoting agent treated controls.Feeding RNA Extracts from Insects that Produce dsRNA

RNA was extracted from a group of 100 flies that had been injected asembryos with the phspGUS[i/r] plasmid. The injected embryos had beensubjected to a single heat shock to produce GUS dsRNA during midembryogenesis. As no plasmid DNA could be detected in developmentalstages beyond 1^(st) instars, it is not expected that further RNA wouldbe transcribed from this template DNA. The extracted RNA was injectedinto embryos at a concentration of 1 ug/ul and the embryos were laterassayed for GUS activity. GUS activity was reduced by 40% in theseembryos, which indicates that the dsRNA is both extractable and stillcapable of promoting gene silencing when transferred back into naïveinsects. RNAs obtained from flies previously injected with either thephspGUS[s] plasmid (sense RNA) or phspGUS[a/s] plasmid (antisense RNA)were also injected into embryos, and these embryos showed no change inGUS activity (results not shown).

TABLE 7 Comparison of transfection promoting agent efficiencie atproducing RNAi-induced delayed development in H. armigera. Valuesrepresent the percentage of larvae that survived the first 12 hpost-treatment that reached pupation by day 10. Six replicates of 10larvae were tested for each of the conditions. RNA mixture % pupation byday 10 Buffer only 87 ± 5  RNA + buffer 84 ± 4  RNA + buffer +spermidine 82 ± 6  RNA + buffer + spermidine + DOTAP 73 ± 7  RNA +buffer + spermidine + Lipofectin 69 ± 10 RNA + buffer + spermidine +Lipofectamine 48 ± 16 RNA + buffer + spermidine + Cellfectin 54 ± 9 RNA + buffer + spermidine + DMRIE-C 53 ± 14 RNA + buffer (nospermidine) + Lipofectamine 58 ± 8 

The RNA extracted from flies previously injected with the phspGUS[i/r]plasmid was then mixed with DMRIE-C and fed to neonate larvae. Developedlarvae and adults were assayed for GUS activity, and 30% of the 3^(rd)instar larvae and 20% of the adults showed between 25 and 50% reductionin GUS activity (FIG. 6). These results indicate that dsRNA can be fedto neonates not only as in vitro transcribed and annealed full lengthinverted repeat dsRNA, but also as dsRNA that has been processed withinthe insect. Although the proportion of dsRNA relative to the total RNAextracted was not determined, the quantity of dsRNA extracted from theinsects was obviously sufficient to promote gene silencing in the fedneonates.

Discussion

The present inventors have demonstrated that dsRNA can be delivered toarthropods. Direct feeding of naked, unpackaged, dsRNA failed to producean RNAi phenotype in D. melanogaster or H. armigera, indicating that thetransfection promoting agents were necessary for effective transfectionin these species. However, it is envisaged that in arthropods with asimple digestive system naked dsRNA may be affective in obtaining genesilencing.

Notably, the same transfection promoting agents were effective atdelivering dsRNA in D. melanogster and H. armigera, despite the pH todifferences in the guts of these two species.

A significant finding was that dsRNA that had been previously processedwithin one arthropod could still facilitate RNAi in another arthropod,even when the RNA was purified from its associated proteins. It isanticipated that the purification process would remove alldsRNA-associated proteins, such as the so-called dicer proteins, whichare believed to mediate target RNA degradation. Assuming that themajority of the dsRNA purified from the arthropods, and subsequentlyingested by the neonates was the processed 21- and 22-meroligonucleotides, it appears that the effective functional unit in thelatter experiment is the short oligonucleotides. However, longer lengthsof dsRNA are clearly effective once ingested, as evidenced by theingestion of in vitro transcribed GUS and vATPase dsRNAs.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed, particularly inAustralia, before the priority date of each claim of this application.

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1. A method of reducing the level of a target RNA in a coleopteraninsect comprising feeding to the insect in a larval stage a compositioncomprising a dsRNA molecule and phosphatidylcholine, wherein the dsRNAmolecule specifically reduces the level of the target RNA in a cell ofthe insect.
 2. The method of claim 1, wherein production of a proteinencoded by the target RNA is reduced.
 3. The method of claim 1, whereinthe method comprises wholly or partially soaking the insect in thecomposition comprising the dsRNA.
 4. The method of claim 3, wherein thecomposition further comprises a nucleic acid condensing agent.
 5. Themethod of claim 4, wherein the nucleic acid condensing agent is selectedfrom the group consisting of: spermidine and protamine sulfate.
 6. Themethod of claim 1, wherein the dsRNA is from a transgenic organismexpressing the dsRNA.
 7. The method of claim 6, wherein the transgenicorganism is a transgenic plant.
 8. The method of claim 1, wherein thedsRNA comprises a nucleotide sequence having at least 90% identity to atleast a portion of the sequence of the target RNA.
 9. The method ofclaim 1, wherein the dsRNA molecule comprises 21 contiguous nucleotidesin a sequence identical to the sequence of a portion of the target RNA.10. The method of claim 1, wherein the dsRNA confers lethality on thecoleopteran insect.
 11. The method of claim 9, wherein the portion ofthe dsRNA which is double stranded is 21 to 50 base pairs in length. 12.The method of claim 1, wherein the dsRNA is partially double-stranded.13. The method of claim 1, wherein the dsRNA is formed by a singleself-complementary RNA strand.
 14. The method of claim 13, wherein theself-complementary RNA strand has a region of self-complementarity ofwhich the sense sequence consists of 20 to 23 contiguous nucleotidesidentical in sequence to 20 to 23 contiguous nucleotides of the targetRNA.
 15. The method of claim 1, wherein the dsRNA is formed by twocomplementary RNA strands.
 16. The method of claim 15, wherein theportion of the dsRNA which is double stranded is 21 to 23 basepairs inlength.
 17. The method of claim 16, wherein the dsRNA is fullydouble-stranded.
 18. The method of claim 1, wherein the effect on thecoleopteran insect is death or sterility.
 19. A method of reducing thelevel of an RNA in a coleopteran insect comprising delivering to acoleopteran insect larva a composition comprising a dsRNA molecule andphosphatidylcholine, wherein the composition is ingested by the larva,and wherein the dsRNA molecule comprises 21 contiguous nucleotides in asequence identical to the sequence of a portion of the RNA in the larva.20. The method of claim 19, wherein the portion of the dsRNA which isdouble stranded is 21 to 23 basepairs in length.